1. Field of the Invention
This invention relates to a method for immunological assay of immunologically active substances in vitro using a multilayer analysis sheet. More particularly, this invention relates to a method for immunological assay using a multilayer analysis sheet comprising a support having provided thereon (1) a silver halide photosensitive layer and (2) a layer capable of separating an antigen or antibody from an antigen-antibody bound product, a so-called B/F separating layer, in which silver halide is utilized in detecting such immunologically active substances. In this invention, this particular method is tentatively termed "photochemical immunological assay".
2. Brief Description of the Prior Art
Since Berson and Yalow succeeded in determining insulin in blood serum using bovine insulin labelled with radioactive iodine and anti-insulin antibody found in serum of a patient with diabetes in 1958, radioimmunoassay (referred to as "RIA") has widely been used. However, various shortcomings are encountered with RIA because radioactive substances are used. For this reason, attention has recently be brought to assays in which a radioisotope labelling is replaced by other labellings. Thus, enzyme immunoassay, fluorescent immunoassay and the like have been proposed, in which enzyme, fluorescent substances, etc. are used, respectively, as labelling substances.
RIA, enzyme immunoassay, fluorescent immunoassay, etc., however, involve disadvantages, on the other hand, that a centrifugal separator should be used upon B/F separation, time required for assay is prolonged and procedures become complicated. For example, the following methods for B/F separation are established in RIA, i.e., a paper filter method (R. S. Yalow and S. A. Berson, J. Clin. Invest., 39, 1157 (1960)), a two antibody method (C. R. Morgan and A. Lazarow, Diabetes, 12, 115 (1963)), a salting-out method (G. M. Grodsky and P. H. Forsham, J. Clin. Invest., 39, 1070 (1960)), an ethanol precipitation method (Nakamura et al.: TONYOBYO (DIABETES), 15, 18 (1972)), a polyethylene glycol method (Nakagawa et al., TONYOBYO (DIABETES), 15, 403 (1972)), a dextran carbon powder method (V. Herbert et al., J. Clin. Endocr., 25, 1375 (1965)), a resin method (F. Melani et al., Klin. Wschr., 43, 1000 (1965)), a talc method (G. Rosselin et al., NATURE 212, 355 (1966)), a Sephadex method (Ide, RINSHO SEIJINBYO, 2, 87 (1972)), etc. Any of these methods possesses both advantages and disadvantages.
However, RIA is subject to several disadvantages due to the use of radioactive isotopes. Thus, RIA involves the danger of radiation exposure and it is necessary to manage expensive and unstable isotopes which can result in reduced accuracy of assay. In addition, special installations, equipment and personnel qualified to deal with radiation are required. Finally, after RIA, disposal of radioactive waste material and the ensuing pollution problems are encountered.
Thus, the photochemical immunological assay has previously been proposed, details of which are disclosed in our co-pending applications Ser. Nos. 126,919 and 126,920, both filed Mar. 3, 1980.